Recombinant human albumin-interleukin-11 fusion protein with long-lasting biological effects

ABSTRACT

Compositions, kits and methods are provided for promoting general health or for prevention or treatment of diseases by using novel recombinant fusion proteins of human serum albumin (HSA) and bioactive molecules. The bioactive molecules may be a protein or peptide having a biological function in vitro or in vivo, and preferably, having a therapeutic activity when administered to a human. By fusing the bioactive molecule to HSA, stability of the bioactive molecule in vivo can be improved and the therapeutic index increased due to reduced toxicity and longer-lasting therapeutic effects in vivo. In addition, manufacturing processes are provided for efficient, cost-effective production of these recombinant proteins in yeast.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation Application of patent applicationSer. No. 11/825,686, filed Jul. 8, 2007, now issued as U.S. Pat. No.7,442,371 B2, which is a Continuation Application of patent applicationSer. No. 10/609,346, filed Jun. 26, 2003, now issued as U.S. Pat. No.7,244,833 B2, which claims the priority of Provisional Application Ser.No. 60/392,948, filed Jul. 1, 2002. All parent applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the manufacture and use of recombinant albuminfusion proteins and combinations thereof, and particularly to yeastexpressed fusion proteins formed between human albumin and bioactivemolecules such as therapeutic proteins and peptides, and moreparticularly to yeast expressed fusion proteins formed between humanalbumin and cell proliferation stimulatory factor (CPSF), such as, bloodcell-stimulatory factors, erythropoietin (EPO), interleukins (ILs), stemcell factor (SCF), thrombopoietin (TPO), granulocyte colony stimulatingfactor (G-CSF), and granulocyte macrophage colony stimulating factor(GM-CSF).

2. Description of Related Art

1. Albumin

Albumin is a soluble, monomeric protein which comprises about one-halfof the blood serum protein. Albumin functions primarily as a carrierprotein for steroids, fatty acids, and thyroid hormones and plays a rolein stabilizing extracellular fluid volume. Mutations in this gene onchromosome 4 result in various anomalous proteins. Albumin is a globularun-glycosylated serum protein of molecular weight 65,000. The humanalbumin gene is 16,961 nucleotides long from the putative ‘cap’ site tothe first poly(A) addition site. It is split into 15 exons which aresymmetrically placed within the 3 domains that are thought to havearisen by triplication of a single primordial domain. Albumin issynthesized in the liver as pre-pro-albumin which has an N-terminalpeptide that is removed before the nascent protein is released from therough endoplasmic reticulum. The product, proalbumin, is in turn cleavedin the Golgi vesicles to produce the secreted albumin. HSA has 35cysteins; in blood this protein monomer has 17 disulfide linkage (Brown,J. R. “Albumin structure, Function, and Uses” Pergamon, New York, 1977).HSA is misfolded when produced intracellularly in yeast without itsamino terminal secretion peptide sequence. This conclusion is based onits insolubility, loss of great than 90% of its antigenicity (ascompared to human-derived HSA), and formation of large proteinaggregates. At present albumin for clinical use is produced byextraction from human blood. The production of recombinant albumin inmicroorganisms has been disclosed in EP 330 451 and EP 361 991.

Albumin is a stable plasma transporter function provided by any albuminvariant and in particular by human albumin. HSA is highly polymorphicand more than 30 different genetic alleles have been reported (WeikampL, R, et al., Ann. Hum. Genet., 37 219-226, 1973). The albumin molecule,whose three-dimensional structure has been characterized by X-raydiffraction (Carter D. C. et al., Science 244, 1195-1198, 1989), waschosen to provide the stable transporter function because it is the mostabundant plasma protein (40 g per liter in human), it has a high plasmahalf-life (14-20 days in human, Waldmann T. A., in “Albumin Structure,Function and Uses”, Rosenoer V. M. et al (eds), Pergamon Press, Oxford,255-275,1977), and above all it has the advantage of being devoid ofenzymatic function, thus permitting its therapeutic utilization at highdose.

2. Interleukin-11 (IL-11)

Human IL-11 (Paul et al. (1990), Pro. Natl. Acad. Sci. 87:7512) has beenidentified in medium conditioned by primate bone marrow-derived stromalcells. IL-11 is expressed in cells of mesenchymal origin, such asstromal fibroblasts, fetal lung fibroblasts and trophoblasts.

IL-11, also called adipogenesis inhibitory factor (AGIF), acts onhematopoietic progenitor cells and stromal cells (Kawashima, I., et al.,Progress in Growth Factor Research, 4,191 1992). The mature molecule isa non-glycosylation protein, 178aa in length, and has an apparentmolecular weight 23 KD (as determined by SDS-PAGE). Human IL-11 geneconsists of five exons and four introns and was mapped on chromosome 19at band 19q13.3-q13.4. IL-11 exhibits a primary structure unrelated tothat of known cytokines, but it often acts similar to other cytokines,notably IL-6. IL-11 is a pleiotropic growth factor effectinghematopoietic and non-hematopoietic cells, often in synergy withinterleukins, colony stimulating factors, or stem cell factor. Inhematopoietic cells, IL-11 can enhance megakaryopoiesis, stimulate earlyand intermediate myeloid progenitor cells, initiate proliferation ofdormant hematopoietic progenitor cells, and stimulate T-cell-dependentdevelopment of antibody-secreting B-cells. In non-hematopoietic cells,IL-11 can inhibit adipogenesis, and mediates the hepatic acute phaseresponse. IL-11 stimulated the production of erythrocytes was reportedonly by Quesniaux, V F J., et al., Blood, 80, 1218 (1992).

Human IL-11 (e.g., NEURMEGA®, manufactured by America Home ProductsCompany) has been approved for clinical trials in the United States fordirectly stimulating the proliferation of hematopoietic stem cells andmegakaryocyte progenitor cells and inducing megakaryocyte maturation,resulting in increased platelet production. It has been used for theprevention of severe thrombocytopenia and the reduction of the need forplatelet transfusion following myelosuppressive chemotherapy.

3. Erythropoietin (EPO)

Erythropoietin (EPO) is a glycoprotein that is the principle regulatorof red blood cells growth and differentiation (U.S. Pat. No. 5,547,933).Erythropoiesis, the production of red blood cells, occurs continuouslythroughout the human life span to offset cell destruction.Erythropoiesis is a very precisely controlled physiological mechanismenabling sufficient numbers of red blood cells to be available in theblood for proper tissue oxygenation, but not so many that the cellswould impede circulation. The formation of red blood cells occurs in thebone marrow and is under the control of the hormone EPO.

EPO is an acidic glycoprotein (˜30,400 Daltons) produced primarily bythe kidney and is the principal factor regulating red blood cellproduction in mammals. Renal production of EPO is regulated by changesin oxygen availability. Under conditions of hypoxia, the level of EPO inthe circulation increases and this leads to increased production of redblood cells. The over-expression of EPO may be associated with certainpathophysiological conditions. Polycythemia exists when there is anoverproduction of red blood cells (RBCs). Primary polycythemias, such asPolycythemia vera, are caused by EPO-independent growth of erythrocyticprogenitors from abnormal stem cells and low to normal levels of EPO arefound in the serum of affected patients.

On the other hand, various types of secondary polycythemias areassociated with the production of higher than normal levels of EPO. Theoverproduction of EPO may be an adaptive response associated withconditions that produce tissue hypoxia, such as living at high altitude,chronic obstructive pulmonary disease, cyanotic heart disease, sleepapnea, high-affinity hemoglobinopathy, smoking, or localized renalhypoxia. In other instances, excessive EPO levels are the result ofproduction by neoplastic cells. Cases of increased EPO production anderythrocytosis have been recorded for patients with renal carcinomas,benign renal tumors, Wilms' tumors, hepatomas liver carcinomas,cerebellar hemangioblastomas, adrenal gland tumors, smooth muscletumors, and leiomyomas.

Deficient EPO production is found in conjunction with certain forms ofanemias. These include anemia of renal failure and end-stage renaldisease, anemias of chronic disorders [chronic infections, autoimmunediseases, rheumatoid arthritis, AIDS, malignancies], anemia ofprematurity, anemia of hypothyroidism, and anemia of malnutrition. Manyof these conditions are associated with the generation of IL-1 and TNF-,factors that have been shown to be inhibitors of EPO activity. Otherforms of anemias, on the other hand, are due to EPO-independent causesand affected individuals show elevated levels of EPO. These formsinclude aplastic anemias, iron deficiency anemias, thalassemias,megaloblastic anemias, pure red cell aplasias, and myelodysplasticsyndromes.

The amount of erythropoietin in the circulation is increased underconditions of hypoxia when oxygen transport by blood cells in thecirculation is reduced. Hypoxia may be caused by loss of large amountsof blood through hemorrhage, destruction of red blood cells byover-exposure to radiation, reduction in oxygen intake due to highaltitudes or prolonged unconsciousness, or various forms of anemia. Inresponse to tissues undergoing hypoxic stress, erythropoietin willincrease red blood cell production by stimulating the conversion ofprimitive precursor cells in the bone marrow into proerythroblasts whichsubsequently mature, synthesize hemoglobin and are released into thecirculation as red blood cells. When the number of red blood cells incirculation is greater than needed for normal tissue oxygenrequirements, erythropoietin in circulation is decreased.

EPO may occur in three forms: alpha-, beta and asialo-EPO. The alpha-and beta-forms differ slightly in carbohydrate components, but have thesame potency, biological activity and molecular weight. The asialo-formis an alpha- or beta-form with the terminal carbohydrate (sialic acid)removed. EPO is present in very low concentrations in plasma when thebody is in a healthy state wherein tissues receive sufficientoxygenation from the existing number of erythrocytes. This normal lowconcentration is enough to stimulate replacement of red blood cellswhich are lost normally through aging. See generally for references,Testa, et al., Exp. Hematol., 8(Supp. 8), 144-152 (1980); Tong, et al.,J. Biol. Chem., 256(24), 12666-12672 (1981); Goldwasser, J. Cell.Physiol., 110(Supp 1), 133-135 (1982); Finch, Blood, 60(6), 1241-1246(1982); Sytowski, et al., Exp. Hematol., 8(Supp 8), 52-64 (1980):Naughton, Ann. Clin. Lab. Sci., 13(5), 432-438 (1983); Weiss, et al.,Am. J. Vet. Res., 44(10), 1832-1835 (1983); Lappin, et al., Exp.Hematol., 11(7), 661-666 (1983); Baciu, et al., Ann. N.Y. Acad. Sci.,414, 66-72 (1983); Murphy, et al., Acta. Haematologica Japonica, 46(7),1380-1396 (1983); Dessypris, et al., Brit. J. Haematol, 56, 295-306(1984); and, Emmanouel, et al., Am. J. Physiol., 247 (1 Pt 2), F168-76(1984).

Because EPO is essential in the process of red blood cell formation, thehormone has potential useful application in both the diagnosis and thetreatment of blood disorders characterized by low or defective red bloodcell production. See, generally, Pennathur-Das, et al., Blood, 63(5),1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol./Oncol., 4, 191-196,(1982) relating to erythropoietin in possible therapies for sickle celldisease, and Eschbach, et al. J. Clin. Invest., 74(2), pp. 434-441,(1984), describing a therapeutic regimen for uremic sheep based on invivo response to erythropoietin-rich plasma infusions and proposing adosage of 10 U EPO/kg per day for 15-40 days as corrective of anemia ofthe type associated with chronic renal failure. See also, Krane, HenryFord Hosp. Med. J., 31(3), 177-181 (1983).

Prior attempts to obtain erythropoietin in good yield from plasma orurine have proven relatively unsuccessful. Complicated and sophisticatedlaboratory techniques are necessary and generally result in thecollection of very small amounts of impure and unstable extractscontaining erythropoietin. Genetically engineered EPO (U.S. Pat. No.5,547,933) has been expressed in Chinese Hamster Ovary cell line (CHO).It has been approved for administration on clinical trials by FDA andnow manufactures by Amgen Inc. under the name of EPOGEN.

It has been estimated that the availability of erythropoietin inquantity would allow for treatment each year of anemias of 1,600,000persons in the United States alone. See, e.g., Morrison, “Bioprocessingin Space—an Overview”, pp. 557-571 in The World Biotech Report 1984,Volume 2:USA, (Online Publications, New York, N.Y. 1984). Recent studieshave provided a basis for projection of efficacy of erythropoietintherapy in a variety of disease states, disorders and states ofhematologic irregularity: Vedovato, et al., Acta. Haematol, 71, 211-213(1984) (beta-thalassemia); Vichinsky, et al., J. Pediatr., 105(1), 15-21(1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet. Gyneacol.,90(4), 304-311 (1983) (pregnancy, menstrual disorders); Haga, et al.,Acta. Pediatr. Scand., 72, 827-831 (1983) (early anemia of prematurity);Claus-Walker, et al., Arch. Phys. Med. Rehabil., 65, 370-374 (1984)(spinal cord injury); Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182(1984) (space flight); Miller, et al., Brit. J. Haematol., 52, 545-590(1982) (acute blood loss); Udupa, et al., J. Lab. Clin. Med., 103(4),574-580 and 581-588 (1984); and Lipschitz, et al., Blood, 63(3), 502-509(1983) (aging); and Dainiak, et al., Cancer, 51(6), 1101-1106 (1983) andSchwartz, et al., Otolaryngol., 109, 269-272 (1983) (various neoplasticdisease states accompanied by abnormal erythropoiesis).

4. Granulocyte Colony Stimulating Factor (G-CSF)

Granulocyte colony stimulating factor (G-CSF) is produced by monocytesand fibroblasts. It stimulating granulocyte colony formation, activatesneutrophils, differentiates certain myeloid leukemic cell lines and is apotent activator of mature granulocytes (Metcalf D., cell, 43, 5, 1985;Groopman, J. E., Cell,. 50, 5 1987);. Nature human G-CSF is a 19.6 KDglycoprotein having 177 amino acids (Souza, L. M et al., Science, 232,62, 1986). Human and murine GCSF share approximately 75% amino acidsequence homology and have biology cross-reactivity (Morstyn, G., andBurgess, A., Cancer Res., 48, 5624, 1988). The biological activity ofrecombinant human G-CSF was measured in a cell proliferation assay usingNFS-60 cells (Shurafuji, N et al., Exp. Hematol., 17, 116, 1989). HumanG-CSF has been brought to the market under the name of NEUPOGEN® byAmgen, Inc.

5. Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)

Granulocyte-Macrophage colony stimulating factor (GM-CSF) inducesmyeloid progenitor cells from bone marrow to from colonies containsmacrophages and granulocytes in semisolid media. GM-CSF also acts uponmature macrophages, eosinophils and nutrophils to stimulate variousfunctional activities (Mazur, E., and Cohen, J., Clin Pharmacol. Ther.,46, 250, 1989; Morstyn, T G., and Burgess. A., Cancer Res., 48, 5624,1988). GM-CSF is an acidic glycoprotein {18-22 KD human (Wong, G., etal., Science, 228, 810, 1986), 23 KD mouse (Metcalf, D., Blood, 67, 257,1986)} which binds to high affinity receptors on GM-CSF sensitive cells.Although human and mouse GMCSF share 54% amino acid sequence homology,their biological actions are species-specific (Metcalf, D., Blood, 67,257, 1986). Other growth factors and CSFs modulate receptor binding oractions of GM-CSF (Nicola, N., Immunol. Today, 8, 134, 1987). Theproliferative activity of human GMCSF is tested in culture using humanTF-1 cells (Kitamura, T., et al., J. cell Physiol., 140, 323, 1989).Human GM-CSF has been brought to the market under the name of LEUKINE®by Immunex, Inc

6. Macrophage Colony Stimulating Factor (M-CSF)

Macrophage Colony Stimulating Factor (M-CSF) is produced by monocytes,fibroblasts and endothelial cells. It stimulates the formation ofmacrophage colonies (Metcalf, D., Blood, 67, 257, 1986), enhancesantibody-dependent cell mediated cytotoxicity by monocytes andmacrophages (Mufson, R. A. et al., Cellular Immunol., 119, 182, 1989),and inhibits bone resorption by osteoclasts (Hattersley, G., et al., J.Cell Physiol., 137, 199, 1988). M-CSF is glycoprotein and appears in afew different molecular weight forms due to variation in glycosylation.The peptide has 159 amino acids (Kawasaki, E. S., et al., Science, 230,291, 1985).

7. Thrombopoietin (TPO)

Thrombopoietin (TPO), the ligand for the receptor encoded by the c-Mplproto-oncogene, acts as a stimulator of the development of megakaryocyteprecursors of platelets. Similar to erythropoietin, TPO leads to anincrease in number of circulating platelets. TPO affects the entirethrombopoietic process, with stronger effects in the later stages. Otherthrombopoietic cytokines include Stem cell factor (SCF), IL-3, IL-6 andIL-11.

TPO is an approximately 35 KD polypeptide of 335 amino acid. However,due to glycosylation the protein has an apparent molecular weight of 75KD in SDS-PAGE. The precursor form of TPO consists of 356 amino acids.To generate the mature TPO (335aa), the precursor cleaves a 21 aminoacids signal peptide. Human, mouse and dog TPO shows 69-75% amino acidhomology. The biological activities of recombinant human TPO wasmeasured in a cell proliferation assay using MO7e cells.

8. Interleukin-3 (IL-3)

Interleukin -3 (IL-3) is one of a large and growing group of growthfactors which support the proliferation and differentiation ofhematopoietic progenitors as well as cells committed to various myeloidlineages in vitro and in vivo. Human IL-3 has 133 amino acids in matureprotein and the glycosylation is not necessary for biological activityin vitro and in vivo. The homology between human and murine IL-3 isconsiderably less. The initial studies on the biology and biochemistryof IL-3 shows that among the well characterized hematopoietic growthfactors, IL-3, is the only factor to be predominantly, if notexclusively, produced by activated T cell in normal cells in mice (Iheleand Weinstein, 1986) as well as in human (Yang and Clark 1998). Thestructure of IL-3, and the structure and location of its gene, are verymuch like those of a number of the hematopoietic growth factors andsuggest that IL-3 is a member of an evolutionarily related family ofgrowth factors. In preclinical and clinical trials, the most prominentand consistent effect of Il-3 in vivo is a significant increase in theabsolute neutrophil count (ANC). In vitro IL-3, in combination withother cytokines such as stem-cell factor, IL-6, IL-1, IL-11, G-CSF.GM-CSF, erythropoietin (EPO), or Thrombopoietin (TPO) induces theproliferation of colony-forming units granulocyte-macrophage (CFU-GM),CFU-Eo, CFU-Baso, burst-forming units-erythroid (BFU-E), colony-formingunits-megakaryocyte (CFU-MK) and colony-formingunits-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) insemisolid medium, and it stimulates the proliferation of purified CD34+cells in suspension culture (Eder, et al., Stem Cell, 15:327-333, 1997).

SUMMARY OF THE INVENTION

The present invention provides compositions, kits and methods forpromoting general health or for prevention or treatment of diseases byusing novel recombinant fusion proteins of human serum albumin (HSA) andbioactive molecules. The bioactive molecules may be a protein or peptidehaving a biological function in vitro or in vivo, and preferably, havinga therapeutic activity when administered to a human. It is believed thatby fusing the bioactive molecule to HSA, stability of the bioactivemolecule in vivo may be improved and the therapeutic index increased dueto reduced toxicity.

In one aspect of the invention, recombinant fusion proteins of humanserum albumin (HSA) and a cell proliferation stimulatory factor (CPSF)are provided 1) to stimulate proliferation of multiple cell types,especially cells of various developmental lineages in the blood, 2)allow a slower release of the HSA-CPSF fusion in the body to maximizethe therapeutic effects of the CPSF, and/or 3) to reduce potential sideeffects or toxicity associated with administration of CPSF alone. Inaddition, manufacturing processes are provided for efficient,cost-effective production for producing these recombinant proteins inyeast.

In another aspect of the invention, an isolated polynucleotide isprovided that encodes a fusion protein formed between HSA and a CPSF,i.e., an HSA/CPSF fusion. The CPSF may include any protein that canstimulate cell proliferation and/or production, preferably selected fromthe group consisting of colony-stimulatory factors such ascolony-stimulating factors such as G-CSF, GM-CSF, eosinophil (EOS)-CSF(i.e. Interleukin-5), macrophage (M)-CSF (CSF-1), multi-CSF(i.e. IL-3)and erythropoietin (EPO); interleukins such as IL-1; IL-2; IL-4; IL-6;IL-7; IL-9; IL-10; IL-11; IL-12; IL-13 and IL-18; Steel factors (SLF:c-kit ligand; Stem-cell factor (SCF); mast cell growth factor);erythroid potentiating activity (EPA), Lactoferrin (LF), H-subunitferritin (i.e., acidic isoferritin), prostaglandin (PG) E1 and E2, tumornecrosis factor (TNF)-α, -β (i.e. lymphotoxin), interferon (IFN)-α (1b,2a and 2b), -β, -ω and -γ; transforming growth factor (TGF)-β, activin,inhibin, leukemic inhibitory factor, oncostatin M; and chemokines suchas macrophage inflammatory protein (MIP)-1-α (i.e. Stem-cell inhibitor);macrophage inflammatory protein (MIP)-1β; macrophage inflammatoryprotein (MIP)-2-α (i.e., GRO-β); GRO-α; MIP-2-β (i.e., GRO-γ); plateletfactor-4; IL-8; macrophage chemotactic and activating factor and IP-10.

The CPSF may be linked directly to the N-terminus or the C-terminus ofHSA to form an HSA-CPSF fusion. Optionally, there is a peptide linker(L) linking HSA and CPSF together to form the fusion protein:HSA-L-CPSF, or CPSF-L-HSA. The length of peptide is preferably between2-100 aa, more preferably between 5-50 aa, and most preferably between14-30 aa. The peptide linker may be a flexible linker that minimizessteric hindrance imposed by the bulky HSA protein on CPSF, such as a(G₄S)₃₋₄ linker.

In one embodiment, an isolated polynucleotide is provided that encodes ahuman serum albumin-interleukin-11 fusion protein (HSA-IL-11). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 1 (FIG. 1). Preferably, the polynucleotide comprises anucleotide sequence at least 95% identical to SEQ ID NO. 1. Preferably,the polynucleotide encodes an amino acid sequence comprising SEQ ID NO.2.

In one embodiment, an isolated polynucleotide is provided that encodes ahuman serum albumin-interleukin-3 fusion protein (HSA-IL-3). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 3. Preferably, the polynucleotide comprises a nucleotidesequence at least 95% identical to SEQ ID NO. 3. Preferably, thepolynucleotide encodes an amino acid sequence comprising SEQ ID NO. 4.

In another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-erythropoietin fusion protein (HSA-EPO).The polynucleotide comprises a nucleotide sequence at least 90%identical to SEQ ID NO. 5. Preferably, the polynucleotide comprises anucleotide sequence at least 95% identical to SEQ ID NO. 5. Preferably,the polynucleotide encodes an amino acid sequence comprising SEQ ID NO.6. [HSA-EPO].

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-granulocyte colony stimulating factorfusion protein (HSA-GCSF). The polynucleotide comprises a nucleotidesequence at least 90% identical to SEQ ID NO.7. Preferably, thepolynucleotide comprises a nucleotide sequence at least 95% identical toSEQ ID NO. 7. Preferably, the polynucleotide encodes an amino acidsequence comprising SEQ ID NO. 8.

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-granulocyte macrophage colony stimulatingfactor fusion protein (HSA-GMCSF). The polynucleotide comprises anucleotide sequence at least 90% identical to SEQ ID NO. 9. Preferably,the polynucleotide comprises a nucleotide sequence at least 95%identical to SEQ ID NO. 9. Preferably, the polynucleotide encodes anamino acid sequence comprising SEQ ID NO. 10.

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-CPSF fusion protein (HSA-CPSF). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 11. Preferably, the polynucleotide comprises a nucleotidesequence at least 95% identical to SEQ ID NO. 11. Preferably, thepolynucleotide encodes an amino acid sequence comprising SEQ ID NO. 12.Optionally, the polynucleotide further comprises a nucleotide sequenceat least 90% identical to SEQ ID NOs. 13, 15, 17, 19, or 21. Preferably,the polynucleotide further comprises a nucleotide sequence encoding anamino acid sequence comprising SEQ ID NOs. 14, 16, 18, 20, or 22.

According to the embodiment, the CPSF may be selected from the groupconsisting of G-CSF, GM-CSF, (EOS)-CSF, CSF-1, EPO, IL-1; IL-2, IL-3,IL-4; IL-6; IL-7; IL-8, IL-9; IL-10; IL-11; IL-12; IL-13, IL-18, SLF,SCF, mast cell growth factor, EPA, Lactoferrin, H-subunit ferritin,prostaglandin (PG) E1 and E2, TNF-α, TNF-β, IFN-α, IFN-β, IFN-ω, IFN-γ;TGF-β, activin, inhibin, leukemic inhibitory factor, oncostatin M,MIP-1-α, MIP-1β; MIP-2-α, GRO-α; MIP-2-β, platelet factor-4, macrophagechemotactic and activating factor and IP-10.

The fusion protein may be a secretory protein, which binds to a specificantibody of human albumin, and optionally, binds to a specific antibodyof the CPSF in this fusion protein.

In another aspect of the invention, a recombinant vector is providedthat comprises the sequence of the polynucleotide described above. Therecombinant vectors can be an expression vector for expressing thefusion protein encoded by the polynucleotide, HSA-CPSF, HSA-L-CPSF, orCPSF-L-HSA in a host organism. The host organism includes, but is notlimited to, mammalian (e.g., human, monkey, mouse, rabbit, etc.), fish,insect, plant, yeast, and bacterium.

In a preferred embodiment, the host organism belongs to a genus of yeastsuch as Saccharomyces (e.g., S. cerevisiae), Pichia, Kluyveromyces,Torulaspora, and Schinosaccharomyces. In a more preferred embodiment,the host organism is Pichia pastoris. In a particular embodiment, therecombinant vector is a pPICZ A, pPICZ B, or pPICZ C.

Depending upon the host organism employed in a recombinant process forproducing the fusion proteins, the recombinant fusion proteins of thepresent invention may be glycosylated or may be non-glycosylated.Preferably, when expressed in a host organism, the fusion protein of HSAand CPSF is glycosylated to substantially the same extent as that whenexpressed in mammalian cells such as Chinese hamster ovarian (CHO)cells, or as that when expressed in Pichia pastoris.

In yet another aspect of the invention, a recombinant cell is providedthat is capable of expressing the sequence of the polynucleotidedescribed above. The recombinant cell may constitutively or be inducedin the presence or absence of an agent to express the fusion proteinencoded by the nucleic acid, HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSA in ahost organism. The type of the recombinant cell includes, but is notlimited to, mammalian (e.g., human, monkey, mouse, rabbit, etc.), fish,insect, plant, yeast, and bacterial cells.

When stored at ambient temperature or a lower temperature, the fusionprotein of HSA and CPSF may have a shelf-life 2 times longer, preferably4 times longer, more preferably 6 times, and most preferably 10 times,longer than that of the CPSF alone (i.e., not fused to HSA) stored underthe same condition.

The fusion protein of the present invention may be administered to amammal, preferably a human, via a variety of routes, including but notlimited to, orally, parenterally, intraperitoneally, intravenously,intraarterially, topically, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery (for exampleby catheter or stent), subcutaneously, intraadiposally,intraarticularly, or intrathecally. The antibody may also be deliveredto the host locally (e.g., via stents or cathetors) and/or in atimed-release manner. In a particular embodiment, the fusion protein isdelivered parenterally via injection.

When delivered in vivo to an animal, the fusion protein of HSA and CPSFmay have a plasma half-life 2 times longer, preferably 4 times longer,more preferably 6 times, and most preferably 10 times, longer than thatof the CPSF alone (i.e., not fused to HSA).

The HSA/CPSF fusion proteins of the present invention may also beadministered in combination with a natural or recombinant human albumin,preferably a recombinant human serum albumin at a therapeuticallyeffective dose and ratio.

In yet another aspect of the invention, combinations of differentHSA/CPSF fusion proteins of are provided. The specific combinations ofthese fusion proteins may be administered to a patient to stimulateproliferation of multiple types of cells in the body or tosynergistically enhance proliferation of a particular cell type.

In one embodiment, HSA/IL-11 fusion may be combined with HSA/EPO fusionand the resulting combination may be administered to a patient with ahematological disorder to simultaneously stimulate proliferation oferythrocytes and platelets.

In another embodiment, HSA/IL-3 fusion may be combined with HSA/EPOfusion and the resulting combination may be administered to a patientwith a hematological disorder to enhance EPO-induced production oferythrocytes.

In yet another embodiment, HSA/IL-3 fusion may be combined with HSA/GCSFfusion and the resulting combination may be administered to a patientwith a hematological disorder to increase the production of erythrocytesand neutrophils, as well as eosinophils.

Alternatively, an HSA/CPSF fusion may be co-administered with adifferent HSA/CPSF fusion simultaneously or sequentially to a patient inneed thereof. This combination therapy may confer synergistictherapeutic effects on the patients.

In yet another aspect of the invention, a method is provided fortreating a patient with a CPSF in need thereof. In one embodiment, themethod comprises: administering a pharmaceutical formulation comprisinga fusion protein of HSA and CPSF to the patient in a therapeuticallyeffective amount. The pharmaceutical formulation may contain anypharmaceutically acceptable excipient and agents that stabilizes theHSA/CPSF fusion protein. The pharmaceutical formulation may furthercomprise natural or recombinant human serum albumin and/or another,different HSA/CPSF fusion protein.

The pharmaceutical formulation may contain any pharmaceuticallyacceptable excipient and agents that stabilizes the HSA/CPSF fusionprotein. The pharmaceutical formulation may further comprise natural orrecombinant human serum albumin and/or another, different HSA/CPSFfusion protein.

In another embodiment, the method comprises: administering a firstpharmaceutical formulation comprising a first fusion protein of HSA anda first CPSF to the patient in a therapeutically effective amount; andadministering to the patient a second pharmaceutical formulationcomprising a second fusion protein of HSA and a second CPSF to thepatient in a therapeutically effective amount. Such a combinationtherapy may confer synergistic therapeutic effects on the patient.

For example, HSA-IL-11 fusion protein may be administered to the patientfirst, followed by administration of HSA-EPO, HSA-GCSF and/or HSA-GMCSFat therapeutically effective doses and ratios to stimulate proliferationof different types of blood cells.

In yet another aspect of the invention, a kit is provided, comprising: afirst fusion protein of HSA and a first CPSF, and a second fusionprotein of HSA and a second CPSF. The first and second CPSFs may be thesame or different. For example, the first CPSF is IL-11 and the secondCPSF is EPO; the first CPSF is IL-3 and the second CPSF is EPO; or thefirst CPSF is IL-11 and the second CPSF is GCSF.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows nucleotide and amino acid sequences of embodiments ofHSA-CPSF fusion proteins, HSA, and examples of individual CPSFs.

FIG. 2 illustrates a plasmid DNA vector contains the HSA sequence and asa backbone vector for making HSA-CPSF fusion proteins.

FIG. 3 shows a Western blot detected using mouse monoclonal anti-humanserum albumin (Sigma Cat# A6684). Each lane was load with equivalent of100 ng of proteins. A), HSA (65 Kd) from blood plasma; B), HSA (65 Kd)from yeast; C), HSA/hG-CSF (84.2 Kd); D), HSA/hEPO (83.5 Kd); E),HSA/hIL-11 (84.5 Kd).

FIG. 4 shows a Western blot detected using goat polyclonal anti-hIL-11antibody (R&D Systems, Cat# Ab-218-NA), each lane contains 100 ngproteins. A), human IL-11 expressed by E. coli; B), HSA/hIL-11 fusionprotein expressed by yeast.

FIG. 5 shows a Western blot detected using monoclonal anti-hGMCSFantibody (R&D Systems), each lane contains 20 ng proteins. A), HumanGMCSF expressed by E. coli; B), HSA/hGMCSF fusion protein expressed inyeast.

FIG. 6 is a bioassay for human IL-11 and HSA/hIL-11 fusion protein instimulation of T1165 cell proliferation. A), with hIL-6 antibody inmedium; B), without hIL-6 antibody in medium.

FIG. 7 is an ELISA bioassay for EPO and HSA fusion protein, HSA/hEPO.

FIG. 8 shows the results of a stability test of rHSA/hIL-11 fusionprotein under different temperature and its cell proliferation activity.A), 37° C.; B), 50° C.

FIG. 9 shows the results of an in vivo animal test of synergisticeffects of a combination of different HSA-CPSFs in stimulating multipleblood cell proliferation, as compared with those when single HSA-CPSFsor CPSFs were administered.

FIG. 10 shows a SDS-PAGE of purified HSA-CPSFs. Each lane was loadedwith about 20 μg of protein. A), HSA (65 Kd) from blood plasma; B), HSA(65 Kd) from yeast; C), HSA/hG-CSF (84.2 Kd); D), HSA/hEPO (83.5 Kd);E), HSA/hIL-11(84.5 Kd).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides innovative compositions, kits and methodsfor modulating cell proliferation in vivo and more particularly forpromoting cell growth for enhancing general health or for treatingdiseases or undesirable conditions.

In general, recombinant fusion proteins of human serum albumin (HSA) anda cell proliferation stimulatory factor (CPSF) are provided in order tocircumvent problems associated with conventional therapy using the CPSFprotein itself. Generally, compared with the CPSF protein alone, theinventive fusion proteins of the present invention possess the followingadvantages: 1) being capable of stimulating proliferation of multiplecell types, especially cells of various developmental lineages in theblood; 2) allowing a slower release of the HSA-CPSF fusion in the bodyto maximize the therapeutic effects of the CPSF, and/or 3) reducingpotential side effects or toxicity associated with administration ofCPSF alone.

The present invention also provides a method for treating a patient witha CPSF in need thereof. In one embodiment, the method comprises:administering a pharmaceutical formulation comprising a fusion proteinof HSA and CPSF to the patient in a therapeutically effective amount.The pharmaceutical formulation may contain any pharmaceuticallyacceptable excipient and agents that stabilizes the HSA/CPSF fusionprotein. The pharmaceutical formulation may further comprise natural orrecombinant human serum albumin and/or another, different HSA/CPSFfusion protein. The pharmaceutical formulation may contain anypharmaceutically acceptable excipient and agents that stabilizes theHSA/CPSF fusion protein. The pharmaceutical formulation may furthercomprise natural or recombinant human serum albumin and/or another,different HSA/CPSF fusion protein.

In addition, the present invention also provides manufacturing processesefficient, cost-effective production for producing these recombinantfusion proteins in yeast. In particular, fusion proteins of HSA witheach of human IL-11, EPO, G-GCSF and GM-CSF have been expressed in ayeast strain of Pichia pastoria and shown to have superior stability instorage and in plasma, and when combined, to possess synergistic effectson the production and growth of multiple types of blood cells.

1. HSA/CPSF Fusion Proteins

In one aspect of the invention, isolated polynucleotides are providedthat encode fusion proteins formed between HSA and a CPSF, i.e.,HSA/CPSF fusion. It should be noted other types of albumin can also beemployed to produce a fusion protein with a CPSF of the presentinvention.

The CPSF may include any protein that can stimulate cell proliferationand/or production. In a particular embodiment, the CPSF is ahematopoietically active cytokine Examples of such a CPSF are describedin Aggarwal and Puri (1995) “Role of cytokines in immuno-regulation”, in“Human Cytokines: Their role in disease and therapy”, edited by Aggarwaland Puri, Blackwell Science Inc., Cambridge, Mass., USA, pp 28, which isincorporated herein by reference in its entirety.

Specific examples of the CPSF include, but are not limited to,colony-stimulating factors such as G-CSF, GM-CSF, eosinophil (EOS)-CSF(i.e. Interleukin-5), macrophage (M)-CSF (CSF-1), multi-CSF(i.e. IL-3)and erythropoietin (EPO); interleukins such as IL-1; IL-2; IL-4; IL-6;IL-7; IL-9; IL-10; IL-11; IL-12; IL-13 and IL-18; Steel factors (SLF:c-kit ligand; Stem-cell factor (SCF); mast cell growth factor);erythroid potentiating activity (EPA), Lactoferrin (LF), H-subunitferritin (i.e., acidic isoferritin), prostaglandin (PG) E1 and E2, tumornecrosis factor (TNF)-α, -β (i.e. lymphotoxin), interferon (IFN)-α(1b,2a, and 2b), -β, -ω and -γ, transforming growth factor (TGF)-β, activin,inhibin, leukemic inhibitory factor, oncostatin M; and chemokines suchas macrophage inflammatory protein (MIP)-1-α (i.e. Stem-cell inhibitor);macrophage inflammatory protein (MIP)-1β; macrophage inflammatoryprotein (MIP)-2-α (i.e., GRO-β); GRO-α; MIP-2-β (i.e., GRO-γ); plateletfactor-4; IL-8; macrophage chemotactic and activating factor and IP-10.

Four distinct colony-stimulating factors (CSFs) that promote survivalproliferation and differentiation of bone marrow precursor cells havebeen well characterized: GM-CSF, G-CSF, M-CSF and Interleukin-3 (IL-3,Multi-CSF). Both GM-CSF and IL-3 are multipotent growth factors,stimulating proliferation of progenitor cells from more than onehematopoietic lineage. In contrast, G-CSF and M-CSF arelineage-restricted hematopoietic growth factors, stimulating the finalmitotic divisions and the terminal cellular maturation of partiallydifferentiated hematopoietic progenitors. Erythropoietin is ahematopoietic growth factor that stimulated red blood cellproliferation. Growth factors, such as Stem Cell Factor, all theinterleukins, and all the interferon, all are CPSF.

The CPSF may be linked directly to the N-terminus or the C-terminus ofHSA to form an HSA-CPSF fusion. Optionally, there is a peptide linker(L) linking HSA and CPSF together to form the fusion protein:HSA-L-CPSF, or CPSF-L-HSA. The length of peptide is preferably between2-100 aa, more preferably between 5-50 aa, and most preferably between14-30 aa. The peptide linker may be a flexible linker that minimizessteric hindrance imposed by the bulk HA protein on CPSF, such as a(G₄S)₃₋₄ linker. The linker addition may be good for CPSF binds to itsreceptor.

The fusion protein may be a secretory protein, which binds to a specificantibody of human albumin, and optionally, binds to a specific antibodyof the CPSF in this fusion protein.

In one embodiment, an isolated polynucleotide is provided that encodes ahuman serum albumin-interleukin-11 fusion protein (HSA-IL-11). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 1. Preferably, the polynucleotide comprises a nucleotidesequence at least 95% identical to SEQ ID NO. 1. Preferably, thepolynucleotide encodes an amino acid sequence comprising SEQ ID NO. 2.[HSA-IL-11].

In one embodiment, an isolated polynucleotide is provided that encodes ahuman serum albumin-interleukin-3 fusion protein (HSA-IL-3). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 3. Preferably, the polynucleotide comprises a nucleotidesequence at least 95% identical to SEQ ID NO. 3. Preferably, thepolynucleotide encodes an amino acid sequence comprising SEQ ID NO. 4.[HSA-IL-3].

In another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-erythropoietin fusion protein (HSA-EPO).The polynucleotide comprises a nucleotide sequence at least 90%identical to SEQ ID NO. 5. Preferably, the polynucleotide comprises anucleotide sequence at least 95% identical to SEQ ID NO. 5. Preferably,the polynucleotide encodes an amino acid sequence comprising SEQ ID NO.6 [HSA-EPO].

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-granulocyte colony stimulating factorfusion protein (HSA-GCSF). The polynucleotide comprises a nucleotidesequence at least 90% identical to SEQ ID NO. 7. Preferably, thepolynucleotide comprises a nucleotide sequence at least 95% identical toSEQ ID NO. 7. Preferably, the polynucleotide encodes an amino acidsequence comprising SEQ ID NO. 8 [HSA-GCSF].

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-granulocyte macrophage colony stimulatingfactor fusion protein (HSA-GMCSF). The polynucleotide comprises anucleotide sequence at least 90% identical to SEQ ID NO. 9. Preferably,the polynucleotide comprises a nucleotide sequence at least 95%identical to SEQ ID NO. 9. Preferably, the polynucleotide encodes anamino acid sequence comprising SEQ ID NO. 10 [HSA-GMCSF].

In yet another embodiment, an isolated polynucleotide is provided thatencodes a human serum albumin-CPSF fusion protein (HSA-CPSF). Thepolynucleotide comprises a nucleotide sequence at least 90% identical toSEQ ID NO. 11. Preferably, the polynucleotide comprises a nucleotidesequence at least 95% identical to SEQ ID NO. 11. Preferably, thepolynucleotide encodes an amino acid sequence comprising SEQ ID NO. 12.Optionally, the polynucleotide further comprises a nucleotide sequenceat least 90% identical to SEQ ID NOs. 13, 15, 17, 19, or 21. Preferably,the polynucleotide further comprises a nucleotide sequence encoding anamino acid sequence comprising SEQ ID NOs. 14, 16, 18, 20, or 22.

According to the embodiment, the CPSF may be selected from the groupconsisting of G-CSF, GM-CSF, (EOS)-CSF, CSF-1, EPO, IL-1; IL-2, IL-3,IL-4; IL-6; IL-7; IL-8, IL-9; IL-10; IL-11; IL-12; IL-13, IL-18, SLF,SCF, mast cell growth factor, EPA, Lactoferrin, H-subunit ferritin,prostaglandin (PG) E1 and E2, TNF-α, TNF-β, IFN-α, IFN-β, IFN-ω, IFN-γ;TGF-β, activin, inhibin, leukemic inhibitory factor, oncostatin M,MIP-1-α, MIP-1β; MIP-2-α, GRO-α; MIP-2-β, platelet factor-4, macrophagechemotactic and activating factor and IP-10.

The above-described polynucleotide with a sequence having a certaindegree of sequence identity, for example at least 95% “identical” to areference nucleotide sequence encoding a HSA/CPSF fusion protein, isintended that the polynucleotide sequence is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence encoding the HSA/CPSF fusion protein. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thepolynucleotide sequence encoding a HSA/CPSF fusion protein can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981),to find the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

When stored at ambient temperature or a lower temperature, the fusionprotein of HSA and CPSF may have a shelf-life 2 times longer, preferably4 times longer, more preferably 6 times, and most preferably 10 times,longer than that of the CPSF alone stored under the same condition.

The present invention involves the utilization of albumin as a vehicleto carry a therapeutic protein such as a CPSF that can be used in thetreatment of certain diseases such as cancers, or people in need of anincreased blood cell proliferation in order to increase the blood cellnumbers. The fusion protein of the present invention may be administeredto a mammal, preferably a human, via a variety of routes, including butnot limited to, orally, parenterally, intraperitoneally, intravenously,intraarterially, topically, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery (for exampleby catheter or stent), subcutaneously, intraadiposally,intraarticularly, or intrathecally. The HSA-CPSF may also be deliveredto the host locally (e.g., via stents or cathetors) and/or in atimed-release manner. In a particular embodiment, the fusion protein isdelivered parenterally via injection.

When delivered in vivo to an animal, the fusion protein of HSA and CPSFmay have a plasma half-life 2 times longer, preferably 4 times longer,more preferably 6 times, and most preferably 10 times, longer than thatof the CPSF alone.

The HSA/CPSF fusion proteins of the present invention may also beadministered in combination with a natural or recombinant human albumin,preferably a recombinant human serum albumin at a therapeuticallyeffective dose and ratio.

It is believed that after fusion with albumin, the CPSF protein can havea longer shelf-life and plasma half-life, which allows cost-effectivestorage and transportation, as well as reduces amount and/or frequencyof drug administration.

It is noted that the same strategy may be used to develop stable fusionproteins with anticancer functions. For example, tumor suppressors, suchas p53, pRb, pRb2/p130, and KLF6 may be fused with HSA. Optionally,insulin and insulin-like growth factor may be fused with HSA fortherapeutic treatment of diabetes. The gene of antibody from human orother sources with therapeutic function also can be fused with albuminfor easy delivery and therapeutic purpose. Antigens against whichvaccines are developed can also be fused to albumin for in vivo deliveryand increasing the antigenicity of the antigens. Antibody can be fusedto albumin and then delivered into blood system to induce an immuneresponse to the invaded foreign antigens such as bacteria, parasites andviruses. In addition anti-aging proteins may be fused with albumin anddelivered to humans.

2. Expression of Fusion Proteins in Host Organisms

The polynucleotides encoding the inventive HSA/CPSF fusion proteins canbe cloned by recombinant techniques into vectors which are introduced tohost cells where the fusion proteins can be expressed.

Generally, host cells are genetically engineered (transduced ortransformed or transfected) with the vectors of this invention which maybe, for example, a cloning vector or an expression vector. The vectormay be, for example, in the form of a plasmid, a viral particle, aphage, etc. The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying the polynucleotides encodingHSA/CPSF fusion proteins. The culture conditions, such as temperature,pH and the like, are those previously used with the host cell selectedfor expression, and will be apparent to the ordinarily skilled artisan.

According to the invention, a recombinant vector is provided thatcomprises the polynucleotide sequence encoding an HSA/CPSF fusionprotein. The recombinant vectors can be an expression vector forexpressing the fusion protein encoded by the nucleic acid, HSA-CPSF,HSA-L-CPSF, or CPSF-L-HSA in a host organism. The host organismincludes, but is not limited to, mammalian (e.g., human, monkey, mouse,rabbit, etc.), fish, insect, plant, yeast, and bacterium.

Expression of the polynucleotide encoding an HSA/CPSF fusion protein isunder the control of a suitable promoter. Suitable promoters which maybe employed include, but are not limited to, adenoviral promoters, suchas the adenoviral major late promoter; or heterologous promoters, suchas the cytomegalovirus (CMV) promoter; the respiratory syncytial virus(RSV) promoter; inducible promoters, such as the MMT promoter, atetracycline or tetracycline-like inducible promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs (including the modified retroviral LTRs hereinabovedescribed); the β-actin promoter; and human growth hormone promoters.The promoter also may be the native promoter which controls thepolynucleotide encoding an HSA/CPSF fusion protein.

Also according to the invention, a recombinant cell is provided that iscapable of expressing comprises the polynucleotide sequence encoding anHSA/CPSF fusion protein. The recombinant cell may constitutively or beinduced in the presence or absence of an agent to express the fusionprotein encoded by the nucleic acid, HSA-CPSF, HSA-L-CPSF, or CPSF-L-HSAin a host organism. The type of the recombinant cell includes, but isnot limited to, mammalian (e.g., human, monkey, mouse, rabbit, etc.),fish, insect, plant, yeast, and bacterial cell.

In a preferred embodiment, the host organism belongs to a genus of yeastsuch as Saccharomyces (e.g., S. cerevisiae), Pichia, Kluyveromyces,Torulaspora, and Schinosaccharomyces. In a more preferred embodiment,the host organism is Pichia pastoris. In a particular embodiment, therecombinant vector is a pPICZ A, pPICZ B, or pPICZ C.

Depending upon the host employed in a recombinant process for producingthe fusion proteins, the fusion proteins of the present invention may beglycosylated or may be non-glycosylated. Preferably, when expressed in ahost organism, the fusion protein of HSA and CPSF may be glycosylated tosubstantially the same extent as that when expressed in mammalian cellssuch as Chinese hamster ovarian (CHO) cells, or as that when expressedin Pichia pastoris.

As indicated above, the albumin fusion proteins of the present inventionare substantially preferably proteomic and can therefore be generated bythe techniques of genetic engineering. The preferred way to obtain thesefusion proteins is by the culture of cells transformed, transfected, orinfected by vectors expressing the fusion protein. In particular,expression vectors capable of transforming yeasts, especially of thegenus Pichia, for the secretion of proteins will be used.

It is particularly advantageous to express the HSA/CPSF fusion proteinin yeast. Such an expression system allows for production of highquantities of the fusion protein in a mature form, which is secretedinto the culture medium, thus facilitating purification.

The development of yeast genetic engineering has been made possible theexpression of heterologous genes and the secretion of their proteinproducts from yeast. The advantages of protein secretion (export) ofyeast are including but not limited to, high expression level, solubleprotein, corrected folding, easy to scale-up and easy for purification.

The HSA/CPSF fusion protein can be secreted into the media of yeast viaan albumin natural secretion signal. The polypeptide sequence of HSAfusion protein can be preceded by a signal sequence which serves todirect the proteins into the secretory pathway. In a preferredembodiment the prepro-sequence of human albumin is used to secret thefusion protein out of yeast cells into the culture medium. Other secretsignal peptides, such as the native Saccharomyces cerevisiae α-factorsecretion signal, can also be used to make fusion protein of the presentinvention.

Yeast-expressed HSA is soluble and appears to have the same disulfidelinkages as the human-blood derived counterpart. If used as apharmaceutical, which may be potentially used in gram amounts in humans,a recombinant HSA will require a close identity with the natural HSAproduct. Secreting the HSA/CPSF fusion protein into the growth media ofyeast, which is via prepro-amino-terminal processing (no initiatormethionine residue), also circumvents the problems associated withpreparing yeast extracts, such as the resistance of yeast cells tolysis. In addition, the purity of the product can be increased obtainedby placing the product in an environment in which 0.5-1.0% of totalyeast proteins is included and the lacks toxic proteins that wouldcontaminate the product.

In a preferred embodiment, a particular species of yeast Pichia pastorisis used the system for expressing HSA/CPSF fusions of the presentinvention. Pichia pastoris was developed into an expression system byscientists at Salk Institute Biotechnology/Industry Association (SIBA)and Phillips Petroleum for high-level expression of recombinantproteins. The techniques related to Pichia are taught in, for example,U.S. Pat. Nos. 4,683,293, 4,808,537, and 4,857,467.

There are some advantages of using yeast Pichia pastoris to expressionof HSA and HSA fusion proteins than using other systems. Pichia pastorisis a species of yeast genus, Pichia. Pichia has many of advantages ofhigher eukaryotic expression systems such as protein processing, proteinfolding, and posttranslational modification, while being as easy tomanipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier,and less expensive to use than other eukaryotic expression systems suchas baculovirus or mammalian tissue culture, and generally gives higherexpression levels. Pichia has an additional advantage which gives 10 to100-fold higher heterologous protein expression levels. Those featuresmake Pichia very useful as a protein expression system.

Owing to the similarity between Pichia and Saccharomyces, manytechniques developed for Saccharomyces may be applied to Pichia. Theseinclude: transformation by complementation, gene disruption, genereplacement. In addition, the genetic nomenclature used for Sac has beenapplied to Pichia. For example, histidinol dehydrogenase is encoded bythe HIS4 gene in both Sac and Pichia. The Pichia as a methylotrophicyeast is capable of metabolizing methanol as its sole carbon source. Thefirst step in the metabolism of methanol is oxidation of methanol toformaldehyde using molecular oxygen by the enzyme alcohol oxidase. Inaddition to formaldehyde, this reaction generates hydrogen peroxide. Toavoid hydrogen peroxide toxicity, methanol metabolism takes place withina specialized cell organelle, called the peroxisome, which sequesterstoxic by-products away from the rest of the cell. Alcohol oxidase has apoor affinity for O₂, and Pichia compensates by generating large amountsof this enzyme. The promoter regulating the production of alcoholoxidase is the one used to drive heterologous (HSA or HSA fused) proteinexpression in Pichia.

Compared with Saccharomyces cerevisiae, Pichia may have an advantage inthe glycosylation of secreted proteins because it generally does nothyper-glycosylate. Both Saccharomyces and Pichia have a majority ofN-linked glycosylation of the high-mannose type; however, the length ofthe oligosaccharide chains added post-translation ally to proteins inPichia (average 8-14 mannose residues per side chain) is much shorterthan those in Saccharomyces (50-150 mannose residues). Very littleO-linked glycosylation has been observed in Pichia. In addition,Saccharomyces core oligosaccharide have terminal α-1,3 glycan linkageswhereas Pichia does not. It is believed that the α-1,3 glycan linkagesin glycosylated proteins produced from Saccharomyces are primarilyresponsible for the hyper-antigenic nature of those proteins making themparticularly unsuitable for therapeutic use. Although not yet proven,this is predicted to be less of a problem for glycoprotein generated inPichia, because it may resemble the glycoprotein structure of highereukaryotes. Protein expressed as a secreted form for correctly refoldingand easy to purification of HSA and HSA fusion proteins. Watanabe, etal. (2001) “In vitro and in vivo properties of recombinant human serumalbumin from Pichia pastoris purified by a method of short processingtime”, Pharm Res 2001 December:18(12):1775; and Kobayashi, K et al.(1998) “The development of recombinant human serum albumin” Ther Apher,November:2(4):257-62.

There are many expression systems available for expressing in Pichia,such as EasySelect™ Pichia Expression Kit from Invitrogen, Inc. On thisvector, an AOX1 promoter is used to allow methanol-inducible high levelexpression in Pichia and a Zeocin™ resistance as selective market forthe recombinants from the transformation. Promoters (transcriptioninitiation region) are very important in expression of fusion proteinsin this invention.

AOX1 gene promoter is a very strong promoter in yeast system, especiallyin Pichia. Two Alcohol Oxidase Proteins are coded in Pichia for alcoholoxidase—AOX1 and AOX2. The AOX1 gene is responsible for the vastmajority of alcohol oxidase activity in the cell. Expression of the AOX1gene is tightly regulated and induced by methanol to very high levels,typically ≧30% of the total soluble protein in cells grown with methanolas the carbon source. The AOX1 gene has been isolated and a plasmid-boneversion of the AOX1 promoter is used to drive expression of the gene ofinterest encoding the desired heterologous protein (Ellis et al., 1985;Koutz et al., 1989; Tschopp et al., 1987a). While AOX2 is about 97%homologous to AOX1, growth on methanol is much slower than with AOX1.This slow growth on methanol allows isolation of Mut^(s) strains (aox1).Except AOX1 gene promoter, other promoters can also be used to driverHSA fusion gene in yeast. They are including the promoter from, but notlimited to, PGK1, GAPDH, Gal1, Gal10, CYC1, PH05, TRP1, ADH1, or ADH2gene.

The expression plasmid can also take the form of shuttle vectors betweena bacterial host such as E. coli, DH5a from GIBCO/Life Science andyeast; the antibiotic Zeocin are used to be a marker for HSA carriervector in all the examples.

The expression vector contains the polynucleotide of HSA or HSA fusiontherapeutic protein are introduced into yeast according to the protocolsdescribed in the kit from Invitrogen Inc. After selection of transformedyeast colonies, those cells expressing the HSA fusion protein ofinterest are inoculated into appropriate selective medium and thentested for their capacity to secrete the given fusion protein into theextracellular medium. The harvesting of the protein can be conductedduring cell growth for continuous cultures, or at the end of the growthphase for batch cultures. The fusion proteins which are the subject ofthis invention are then further purified from the culture supernatant bymethods which take into account the albumin purification methods andpharmacological activities.

It is noted that other expression systems may also be used to expressrHSA and HSA/CPSF fusion proteins, including but not limited to E. coli,B. Subtitis, Saccharomyces, Kluyverornyces, Candida, Torulopsis,Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, animals, plants, and insect cells.

3. Combination Therapy of HSA/CPSF Fusion Proteins

The present invention also provides combinations of different HSA/CPSFfusion proteins. The specific combinations of these fusion proteins maybe administered to a patient to stimulate proliferation of multipletypes of cells in the body or to synergistically enhance proliferationof a particular cell type. In particular, a combination of human albuminfusions with different hematopoietically active cytokines is used toeffectively promoting proliferation of the multiple blood cells andplatelets. By using a combination of HSA/CPSF fusion proteins targetingthe signal transduction pathways of different types of blood cells,multiple blood functional cell production, such as platelets,erythrocytes and macrophages of white cells, can be increased afteradministration by just one injection.

In the present invention, the albumin's plasma transporter function andthe therapeutic function of the CPSF are integrated into a fusion form.The presence of albumin may confer a superior stability to the CPSF byresisting degradation by proteases in the blood circulation, thussignificantly prolonging the plasma half life of the CPSF. Due to themasking effect of a bulky albumin, a combination of different CPSFsfused with albumin may impose less interference with the biologicalfunction(s) among different albumin fused CPSFs than among a combinationof the “naked” CPSFs. Further, a CPSF fused with albumin may be slowlyreleased in the system over an extensive period of time, therebyreducing the toxicity associated with injection of the CPSF alone inabnormally high concentrations in the body. Such a slow release mode ofaction of the fusion protein combination can significantly reduce theamount and/or frequency of injections of the CPSF, thereby furtherreducing the side effects of CPSFs. Such combinations are particularlyuseful for stimulating multiple blood cell proliferation after or beforethe chemo- or radiation therapy of cancer patients whose tolerance forfrequent, high dose injection of CPSF are seriously compromised.

For example, in animal testing human TPO, stem cell factor (SCF) andIL-3 have shown very strong toxicities in vivo. Due to the severetoxicity in vivo, SCF has been approved by the FDA for use in vitroonly. The limitation on SCF has prevented it from being developed intouseful therapeutics in the clinic. The side effects of IL-3 aredose-dependent. At a dose higher than 5 μg/Kg, side effects have beenobserved in patients treated with IL-3, including fever, rash, fatigue,diarrhea, rigor, musculoskeletal pain, chills, headache, conjunctivitis,edema, chest pain, dyspnea, decrease in platelet counts, increasing inbasophilic counts, marrow fibrosis, and pulmonary edema. Eder, M et al.(1997) “IL-3 in the Clinic”, Stem Cells, 15:327-333.

According to the present invention, HSA fusion protein with this type ofCPSF may remove above limitations by slowly releasing the drug into thepatient's system. In addition, such fusion proteins may be combined witha relatively higher amount of albumin to further reduce the impactresulted from directly injecting the drug into the blood which causes astrong, adverse reaction of the central nervous system.

It is also known that “naked” cytokines (i.e., cytokines not fused toanother protein such as HSA) are quite unstable when stored and have ashort plasma half-life. Clearly, a therapeutic protein with such a weakstability in vivo constitutes a major handicap. In effect, repeatedinjections of the product, which are costly and inconvenient forpatient, or an administration of product by perfusion, become necessaryto attain an efficient concentration in plasma. Due to its extendedplasma half-life and enhanced stability, the HSA/CPSF fusion proteins ofthe present invention and their combinations, e.g., HSA fusions withhIL-11, hEPO, hG-CSF and hGM-CSF, can be used to stimulate theproduction of multiple blood cells in plasma of humans.

In one embodiment, HSA/IL-11 fusion may be combined with HSA/EPO fusionand the resulting combination may be administered to a patient with ahematological disorder to simultaneously stimulate proliferation oferythrocytes and platelets. For example, cancer patients may be injectedwith a combination of HSA/IL-11 and HSA/EPO fusion proteins, before orafter, chemotherapy treatment to avoid blood transfusion and tostimulate proliferation of erythrocytes and platelets.

In another embodiment, HSA/IL-3 fusion may be combined with HSA/EPOfusion and the resulting combination may be administered to a patientwith a hematological disorder to enhance EPO-induced production oferythrocytes.

In yet another embodiment, HSA/IL-3 fusion may be combined with HSA/GCSFfusion and the resulting combination may be administered to a patientwith a hematological disorder to increase the production of erythrocytesand neutrophils, as well as eosinophils.

Alternatively, an HSA/CPSF fusion may be co-administered with adifferent HSA/CPSF fusion simultaneously or sequentially to a patient inneed thereof. This combination therapy may confer synergistictherapeutic effects on the patients. In one embodiment, the method isprovided, comprising: administering a first pharmaceutical formulationcomprising a first fusion protein of HSA and a first CPSF to the patientin a therapeutically effective amount; and administering to the patienta second pharmaceutical formulation comprising a second fusion proteinof HSA and a second CPSF to the patient in a therapeutically effectiveamount. Such a combination therapy may confer synergistic therapeuticeffects on the patient.

For example, HSA-IL-11 fusion protein may be administered to the patientfirst, followed by administration of HSA-EPO, HSA-GCSF and/or HSA-GMCSFat therapeutically effective doses and ratios to stimulate proliferationof different types of blood cells.

The present invention further provides a kit for use in the combinationtherapy described above. The kit comprises: a first fusion protein ofHSA and a first CPSF, and a second fusion protein of HSA and a secondCPSF. The first and second CPSFs may be the same or different. Forexample, the first CPSF is IL-11 and the second CPSF is EPO; the firstCPSF is IL-3 and the second CPSF is EPO; or the first CPSF is IL-11 andthe second CPSF is GCSF.

The HSA/CPSF fusion proteins and their combinations thereof may be usedto treat a wide variety of diseases, including but not limited to thehematological disorders such as hypochromia, hypochromic microcyticanemia, and anemia, platelet-less, HIV infection, cancer, renal failure,and tissue/organ transplantation. These fusion proteins are preferrednot to contain non-human sequences that may elicit adverseimmunogenicity in the patient.

EXAMPLES

1. General Molecular Cloning Techniques

The classic methods of molecular cloning including, DNA preparativeextractions, agarose and polyacrylamide electrophoresis, plasmid DNApurification by column or from gel, DNA fragment ligations, andrestriction digestion, are described in detail in Maniatis T. et al.,“Molecular cloning, a Laboratory Manual”, Cold Spring Harbor laboratory,Cold Spring Harbor, N.Y., 1982 and will not be reiterated here.

Polymerase Chain Reaction (PCR) used through out all the examples isdescribed by Saiki, R. K. et al, Science 230:1350-1354, 1985 and iscarried out on a DNA thermal cycler (Perkin Elmer) according to themanufacturer's specification. DNA sequencing was performed by usingstandard facilities and following the method developed by Sanger et al.,Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977. Oligonucleotides weresynthesized by commercial facilities.

Transformation of E. coli was done by using DH5α competent cells fromGIBCO/BRL. Qiagen plasmid DNA purification columns were used in thepurification of plasmid DNAs. The transformation of yeast was carriedout by electroporation following the instruction provided by themanufacturer or according to the manual of EasySelect™ Pichia ExpressionKit (Invitrogen Inc). All yeast stains used in the examples are membersof the family of Pichia, and in particular, the strain of Pichiapastoris (supplied by Invitrogen).

2. Construction of a Backbone Vector Expressing Human Serum Albumin

A total RNA isolated from human fetal liver was used in a reversetranscription polymerase chain reaction (RT-PCR) to generate thepolynucleotide encoding human serum albumin. Briefly, 5 μg of RNA wasreverse transcribed by adding a poly(T)_(18+N) primer and theSuperScript™ II RNase H⁻ reverse transcriptase (GIBCO/BRL) to make thecomplementary first strand of cDNA. The reaction was incubated at 45° C.for 20 minutes, then at 55° C. for 40 minutes.

The primers for cloning human serum albumin (HSA) are the following:

SEQ ID No. 23: 5′-GAATTCATGAAGTGGGTAACCTTTATTTCC-3′ and SEQ ID No. 24:5′-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3′.

These primers were designed based on the HSA sequence published byGenBank (Access# V00494). Two EcoR I (underline of primers) sites werecreated at the 5′ end and 3′ end for sub-cloning into an expressionvector. After inactivating the reverse transcriptase at 94° C. for 4minutes, the DNA encoding of HSA was further amplified by Taq DNA PCR(Perkin Elmer) with 35 cycles of 94° C./30 seconds and 58° C./30 secondsand 72° C./2 minutes 30 second, followed by a 72° C./10 minutesincubation. The PCR product (1842 base pairs) was confirmed by 1%agarose gel electrophoresis. The product was subcloned into a pCR II TAcloning vector from Invitrogen. DNA sequencing confirmed that theplasmid DNA contained an insert whose polynucleotide sequence matchesthe DNA sequence published in GenBank (Access# V00494). FIG. 1, Seq IDNo.11 is a polynucleotide DNA sequence and Seq ID No 12 is the proteinamino acid sequence of human serum albumin.

After restriction digestion of the PCR product with EcoR I, the gelpurified HSA DNA fragment was inserted into a pPICZ-A vector (providedby Invitrogen) at the EcoR I site. After transformation of bacteria DH5αcells with this vector encoding HSA, a colony was selected from a lowsalt LB-agar plate contains 25 μg/ml Zeocin. The direction of the insertwas confirmed by restriction enzyme double digestion of plasmid DNA byXho I/Nde I. The construct was designated as pYZ-HSA (Y: yeast vector;Z: Zeocin resistant) and its physical map is shown in FIG. 2.

There are some advantages associated with the vector constructed above.It confers resistance to the antibiotic Zeocin. Zeocin is isolated fromStreptomyces and is structurally related to bleomycin/phleomycin-typeantibiotics. Antibiotics in the family of bleomycin/phleomycin are broadspectrum antibiotics that act as strong antibacterial and anti-tumordrugs. They show strong toxicity against bacteria, fungi (includingyeast), plants, and mammalian cells. However, Zeocin is not as toxic asbleomycin on fungi. A single antibiotic Zeocin could be used inselecting the recombinants in both bacteria and in yeast. Further, thereare multiple cloning sites at the 3′ end of HSA for convenientlysubcloning a CPSF protein in frame to encode a HSA-CPSF. In addition, amyc epitope sequence and a polyhistidine tag can be fused to theC-terminal of the expressed fusion protein for easy detection and/orpurification by using commercially available antibodies against myc orpolyhistidine tags. This vector, as a backbone vector, was used in theconstruction of expression vectors for all the HSA fusion proteinsdescribed in the Example section.

3. Molecular Cloning of Human IL-11, EPO, G-CSF, and GM-CSF

3.1. Molecular Cloning of Human IL-11 Gene

Human Il-11 was cloned from a total RNA preparation of human bonemarrow-derived stromal cells by RT-PCR method described in Example 2.The oligonucleotide primers are

SEQ ID NO. 25: 5′-CATATGAACTGTGTTTGCCGCCTGGTCC-3′ SEQ ID NO. 26:5′-GATATGTATGACACATTTAATTCCC-3′

A polynucleotide having1051 base pairs (bp) was amplified from RT-PCRreaction and subcloned into pCR II TA cloning vector from InvitrogenInc. DNA sequencing confirmed the reading frame of human IL-11 andinclusion of a 448 by 3′-end un-translation region of hIL-11. An Nde Irestriction enzyme site was created at the 5′ end. The ATG initiatestart codon of hIL-11 was included in this site (underlined in SEQ IDNO. 25). The DNA sequence of hIL-11 (SEQ ID NO. 13) and its amino acidsequence (SEQ ID NO. 14) are shown in FIG. 1.

3.2. Molecular Cloning of Human Erythropoietin Gene

Human erythropoietin gene was obtained by RT-PCR from human fetal kidneymRNA from Clontech Laboratory. EPO specific primers were designed basedon the sequence published by Lin, F K et al., “Cloning and expression ofhuman erythropoietin gene”, Proc. Natl. Acad. Sci. USA.,82(22):7580-7584 (1985). They are

SEQ ID NO. 27: 5′-GGATCCATGGGGGTGCACGAATGTCC-3′, and SEQ ID NO. 28:5′-GAATTCTCATCTGTCCCCTGTCCTGC-3′.

The PCR product was a full length reading frame of EPO with two newlycreated restriction enzymes in each end, Bam HI at the 5′end and EcoR Iat 3′end. The product was inserted into pCR II vector and sequenceconfirmed. The human EPO DNA sequence (SEQ ID NO. 15) and amino acidsequence (SEQ ID NO. 16) are showed in FIG. 1.

3.3. Molecular Cloning of Human G-CSF

Primers used to clone the human G-CSF gene from a cDNA library of humanfetal liver tissues are

SEQ ID NO. 29: 5′-GGATCCATGGCTGGACCTGCCACCC-3′, and SEQ ID NO. 30:5′-GAATTCTCAGGGCTGGGCAAGGTGGC-3′

These primers were designed based on Nagata, S et al., Molecular Cloningand Expression of cDNA for Human Granulocyte Colony-Stimulating Factor,Nature, 319:415-418, 1986. A Bam HI site at 5′end and an EcoR I site at3′end of G-CSF were created. The PCR products were gel-purified andsubcloned into pCR2.1 TA cloning vectors and DNA sequence was confirmed.The human G-CSF DNA sequence (SEQ ID NO. 17) and the amino acid sequence(SEQ ID NO. 18) are shown in FIG. 1.

3.4. Molecular Cloning of Human GM-CSF

Human GM-CSF was cloned from a total RNA sample prepared from humanfetal liver based on Wong, G G et al., “Human GM-CSF: molecular cloningof the complementary DNA and purification of the nature and recombinantproteins” Science, 228:810-815, 1985. The Primers were:

SEQ ID NO. 31: 5′-GGATCCATGTGGCTGCAGAGCCTGCTGC-3′, and SEQ ID NO. 32:5′-GAATTCTCACTCCTGGACTGGCTCC-3′

The PCR products were gel-purified and inserted into pCR2.1 TA cloningvector and sequence confirmed. The human GM-CSF DNA sequence (SEQ ID NO.19) and amino acid sequence (SEQ ID NO. 20) are shown in FIG. 1.

4. In Frame Fusion of HSA with Human IL-11, EPO, G-CSF or GM-CSF

There is a Bsu36 I site at the C′-terminus of HSA. All of the CPSFsdescribed in the Example section were fused into this site by PCR primerextension to generate a restriction enzyme site of Bsu36 I at theN-terminus of the CPSF DNA sequence. The CPSF DNA fragments wereamplified by PCR and then subcloned into Bsu36 I and Xho I sites ofpYZ-HSA vector which had been double digested with Bsu36 I and Xho I tolinearize the plasmid DNA

4.1. Construction of Vector Containing Hybrid Polynucleotide ofHSA/hIL-11

IL-11 gene was fused to HSA C′-terminus by using the following PCRprimers:

SEQ ID NO. 33: 5′-CTGCCTTAGGCTTACCTGGGCCACCACCTGGCC-3′. (Human IL-11mature protein sequence is underlined), and SEQ ID NO. 34:5′-TGTCGACTCACAGCCGAGTCTTCAGCAGC-3′.

A Sal I site (underlined in SEQ ID NO. 34) was created at the 3′ end ofhIL-11 gene because there is a Xho I site in the sequence of matureprotein of IL-11. The Sal I site sequence is a cohesive sequence withXho I site. After ligation, the Sal I and Xho I site were all gone.

The PCR products were digested with Bsu36I and Sal I, and the fragmentwas gel purified and inserted into pYZ-HSA between of Bsu36 I and Xho Isites to generate a new plasmid DNA, pYZ-HSA/hIL-11. The HSA-hIL-11hybrid polynucleotide sequence (SEQ ID NO. 1) and its fusion proteinamino acid sequence (SEQ ID NO. 2) are showed in FIG. 1.

4.2. Construction of Vector Containing Hybrid Polynucleotide of HSA/EPO

To make a HSA-EPO fusion protein, the following primers were designed

SEQ ID NO. 35: 5′-CTGCCTTAGGCTTAATCTGTGACAGCCGAGTCC-3′ (human EPO matureprotein sequence underlined), and SEQ ID NO. 36:5′-CACTCGAGTCATCTGTCCCCTGTCCTGC-3′ (Xho I site underlined)

and used to generate the modified human IL-11 DNA fragment. The PCRproducts were inserted between Bsu36I and Xho I sites of pYZ-HSA togenerate a pYZ-HSA/hEPO. The HSA-EPO hybrid polynucleotide sequence (SEQID NO. 5) and its fusion protein amino acid sequence (SEQ ID NO. 6) areshown in FIG. 1.

4.3. Construction of Vector Containing Hybrid Polynucleotide ofHSA/HG-CSF

Human G-CSF gene was fused with HSA DNA sequence by using two primers:

SEQ ID NO. 37: 5′-CTGCCTTAGGCTTAACCCCCCTGGGCCCTGCCAGC-3′ (G-CSF matureprotein sequence underlined), and SEQ ID NO. 38:5′-CTCGAGTCAGGGCTGGGCAAGGTGG-3′ (Xho I site at the 3′-teminus of G-CSFunderlined).

The PCR products were gel purified and subcloned between Bsu36I and XhoI sites of pYZ-HSA to generate a pYZ-HSA/hG-CSF. The HSA-G-CSF hybridpolynucleotide sequence (SEQ ID NO. 7) and its amino acid sequence (SEQID NO. 8) are shown in FIG. 1.

4.4. Construction of Vector Containing Hybrid Polynucleotide ofHSA/HGM-CSF

The following primers:

SEQ ID NO. 39: 5′-ACTCCTTAGGCTTAGCACCCGCCCGCTCGCCCAGC-3′ (GM-CSF matureprotein sequence underlined), and SEQ ID NO. 40:5′-CTCGAGTCACTCCTGGACTGGCTCC-3′ (Xho I site underlined)

were used to modify GM-CSF DNA sequence in order to subclone it intopYZ-HSA vector. PCR products were gel purified and double digested withBsu36 I and Xho I and inserted between Bsu36 I and Xhol sites of pYZ-HSAto generate a pYZ-HSA/hGMCSF. The HSA/GM-CSF hybrid polynucleotidesequence (SEQ ID NO. 9) and its fusion protein amino acid sequence (SEQID NO. 10) are shown in FIG. 1.

5. Transformation of Yeasts

A yeast Pichia pastoris strain, GS 115, colony was inoculated into 5 mlof YPD medium in a 50 ml conical tube at 30° C. overnight with shakingat 250 rpm. 0.2 ml of the culture was inoculated into 500 ml of YPDmedium continually shaking at 30° C. for further 2-3 hours or until thecell density reach to OD₆₀₀=1.3-1.5. The cells were collected bycentrifuging. The cell pellet resuspend in 500 ml of ice-cold sterilewater in order to wash the cells. After two rounds water washing, thecells were resuspended in 20 ml of ice-cold 1 M sorbitol to wash again.The cells finally suspended in 1 ml of ice-cold 1M sorbitol. The plasmidDNA constructs from Example 2, pYZ-HSA and in Example 4, pYZ-HSA/IL-11,pYZ-HSA/hEPO, pYZ_HSA/hG-CSF, and pYZ-HSA/hGM-CSF was linearized by PmeIrestriction enzyme digestion first.

Five μg of each linear plasmid DNA was used to transform 80 μl of thefreshly made yeast cells in an ice-cold 0.2 cm electroporation cuvette.The cells mixed with plasmid DNA were pulsed for 5-10 ms with fieldstrength of 7500V/cm. After the pulse, 1 ml of ice-cold 1M sorbitol wasimmediately added into the cuvette and the content was transferred to asterile 15 ml tube. The transformed cells were incubated in 30° C.without shaking for 2 hours then spread on pre-made YPD-agar plates with100 μg/ml Zeocin. The colonies were identified with the insert and theexpression level was determined by SDS-PAGE or western-blot with properantibodies.

A consortium of 4 yeast strains produced above (collected referred to asYZ-HSA/CPSFs) of Pichia pastoris encoding HSA/IL-11, HSA/hEPO,HSA/hG-CSF, and HSA/hGM-CSF was deposited to the ATCC® (America TypeCulture Collection) under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. The 4 yeast strains are separately designated asYZ-HSA/IL-11, YZ-HSA/hEPO, YZ-HSA/hG-CSF, and YZ-HSA/hGM-CSF,respectively, and their consortium is deposited at the ATCC® underPatent Deposit Designation No: PTA-4607.

Different strains of Pichia, such as X-33, KM71 and proteinase deficientstrains SMD1168 and ZY101 were tested for the expression and secretoryof recombinant proteins.

6. Secretion and Characterization of HSA-CPSF Fusion Proteins Expressedby Pichia

Several colonies from each transformation of the HSA-CPSF were culturedwith Zeocin in the buffered minimal medium with glycerol overnight oruntil OD₆₀₀=2-6 at 30° C. and shaking at 300 rpm. The cultured cellswere collected by centrifuge at 1500 rpm for 5 minutes. Resuspend thecells into buffered minimal medium without glycerol and cell densitieswas keep in OD₆₀₀=1.0. 100% methanol was added into each flask to afinal concentration at 0.5% every 24 hours to induce the proteinexpression. The culture medium was collected at different time pointsand the expression of each fusion protein was confirmed by SDS-PAGE andwestern blot. The results showed that human albumin and HSA-CPSF fusionprotein were expressed and secreted into the medium.

Mouse monoclonal anti-human serum albumin (Sigma) was used forimmunoblotting on a SDS-PAGE gel. A typical Western blot experiment wascarried on by electrophoresis transfer the protein from SDS-PAG to anylon or nitrocellulose filter and incubated with a specific antibody(as the “first antibody”). Then an anti-first antibody was added to bindto the first antibody (as the “second antibody”). The second antibodywas labeled with Fluorescence and the filter was exposed to an X-rayfilm. Protein molecular weight standard was used to determine theprotein size. The results (FIG. 3) showed that the expressed recombinantproteins, HSA, HSA-CPSF therapeutic fusion protein, had an expectedmolecular weight and also had the same antigen as that of HSA preparedfrom a human blood plasma (Sigma). Using monoclonal anti-hIL-11 specificantibody as the first antibody, the HSA/hIL-11 fusion protein and humanIL-11 (R&D System) had the same antigen and showed that the molar ratioof HSA to hIL-11 in the HSA/hIL-11 fusion protein is as expected (FIG.4). Using monoclonal anti-hGMCSF specific antibody (R&D System) as thefirst antibody, the HSA-GMCSF fusion protein and human GMCSF (R&DSystem) had the same antigen and showed that the molar ratio of HSA toGMCSF in the HSA/GMCSF fusion protein is as expected (FIG. 5).

7. Purification and Molecular Characterization of HSA-CPSF

The cell culture medium (supernatant) containing the secreted protein ofHSA or HSA-CPSF fusion protein produced from the recombinant Pichia wascollected, the salt concentration reduced, and the pH was adjusted toabove 7.5. The concentrated sample was passed through an Affi-GelBlue-gel (50-100mesh) (Bio-Rad). The albumin or albumin fusion proteinwas bound to the matrix and eluded by a gradient 1-5M NaCl. 75-85% purealbumin or albumin-CPSF can be recovered in this step. If furtherpurification is necessary, a size exclusion chromatography is applied togive a 95-99% purity of proteins. The pyrogen was removed from theprotein samples in order to meet the requirement for use in in vivotest. The Affi-Prep Polymyxin Support (BIO-Rad) column was used toremove endotoxin from the samples. The purified protein finally passedthrough 0.2 μM filter to be sterilized and the protein concentration wasmeasured by a standard method by using a Bio-Rad Protein Assay Kit.

8. Cell Proliferation Assay of Human IL-11

A murine plasmacytoma cell line T1165 which is an IL-6 depended cellline was used to carry out a bioassay for IL-11 according to Paul, S Ret al., PNAS 87:7512-7516, 1990. The cells were maintained in RPMImedium supplemented with 10% heat-inactivated fetal calf serum, 2 mMglutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml)(Gibco/BRL), 50 μM 2-meracaptoethanol (sigma), and recombinant humanIL-6 (4-10 ng/ml or 20 U/ml in final concentration) (Gibco/BRL).Bioassay is preformed by 1×10⁴ cells/well were placed into 96-welltissue culture plate in 200 ul of IL-6 free medium for 48 hr in 37° C.in the presence of multiple dilution of hIL-11 purified from Thioredoxinfusion protein in E. coli or from yeast purified HSA/IL-11 fusionprotein. In the final 6 hours, the cells were pulse-labeled with 0.5 mCiof [³H]thymidine (1 Ci=37 GBq, from Dupont) per well. After incubation,the cells were collected on to a glass filter, washed and assayed on aBeckman scintillation counter, Neutralizing goat anti-human IL-6antibody was included to abrogate the effect of IL-6 as control in thetesting of purified protein samples. The control proteins including withor without HSA (from human blood preparation), rHSA expressed in yeast(Pichia) in the lab, Thioredoxin (Trx) was a peptide purified from theenterokinase digested Trx/IL-11 fusion protein. The results are shown inFIG. 6 (A and B panels). The HSA/hIL-11 bioactivities were not affectedby the presence of antibody of human IL-6. HSA fusion protein has about⅓ of cell proliferation activity compared with that human IL-11 alone inthe same amount of protein. Since HSA has a molecular weight about 3times higher than that of hIL-11, it can be inferred that HSA-hIL-11fusion protein has the same bioactivity as that of human IL-11 alone.

9. Bioassay of EPO by ELISA

Enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems was usedfor the quantitative determination of erythropoietin (EPO) concentrationand bioactivities comparison with a commercial EPO sample. The EPO ELISAis based on the double-antibody sandwich method. Microplate wells,precoated with monoclonal (murine) antibody specific for human EPO wereincubated with samples or standard. Erythropoietin binds to theimmobilized antibody on the plate. After removing excess protein sampleor standard, wells were incubated with an anti-EPO polyclonal (rabbit)antibody conjugated to horseradish peroxidase. During the secondincubation, the antibody-enzyme conjugate bound to the immobilized EPO.Excess conjugate was removed by washing. A chromogen was added into thewells and oxidized by the enzyme reaction to form a blue coloredcomplex.

The reaction was stopped by the addition of acid, which turned the blueto yellow. The amount of color generated was directly proportional tothe amount of conjugate bound to the EPO antibody complex, which, inturn, was directly proportional to the amount of EPO in the proteinsamples or standard. The absorbance of this complex was measured and astandard curve was generated by plotting absorbance versus theconcentration of the EPO standards. The EPO concentration of the unknownsample was determined by comparing the optical density of the proteinsamples to the standard curve. The standards used in this assay wererecombinant human EPO (with kit) calibrated against the SecondInternational Reference Preparation (67/343), a urine-derived form ofhuman erythropoietin. Human recombinant EPO expressed in CHO cells wasused as a control to determine the rHSA/EPO bioassay sensitivity.

The results showed that in the bioassay hEPO fused to HSA had 1/10 ofthe sensitivity compared with the standard (FIG. 7). The size of HSA-EPOfusion protein molecule may be too large, which prevents the anti-EPOantibody from efficiently binding to the EPO molecule fused to HSA,thereby reducing the sensitivity of the detection in this bioassay.

10. Stability Testing of HSA-CPSF Fusion Proteins In Vitro

Using HSA/hIL-11 as an example, the stability of this HSA-CPSF fusionprotein was tested at different time points at 37° C. and 50° C. 5 ng ofhuman IL-11 from bacteria or 5 ng of rHSA/hIL-11 was put into 200 μlthin-well PCR tube with 200 μl of tissue culture medium RPM1 withoutfetal bovine serum and other components. The tubes were sealed and leftin water bath. Samples were taken out at different time points andimmediately put into −80° C. for storage. After all of samples werecollected, a cell proliferation test on T1165 cell line was carried outby incorporating ³H-Thymidine into newly synthesized DNA ofproliferating cells. The control of the test was set up in the same wayas that in the bioassay of human IL-11 (See Paul et al., 1990). As shownin FIG. 8, after 5 weeks in 37° C. (Panel A), the bioactivity ofHSA/IL-11 still remained the same, but the “naked” human IL-11 lostalmost all of its bioactivity after three weeks at 37° C. At 50° C.(Panel B), the “naked” human IL-11 lost its the bioactivity completelyin 2 weeks. And the HSA/IL-11 fusion protein still retained at leasthalf of its bioactivity. These results indicate that a CPSF fused tohuman albumin can have a longer storage time and more resistant todegradation in harsh environment such as high temperatures.

11. Synergistic Effects of Combination of HSA-CPSF Fusion Proteins inStimulation of Multicell Proliferation

Rabbits (2.3-2.6 Kg) were injected with 200 μl of recombinant proteinsprepared above at day 1, day 3 and day 6. Rabbit A was injected with amixture of 150 U/kg EPO (about 10 μg of protein) and 100 μg recombinantHSA (rHSA); Rabbit B with a mixture of 120 μg IL-11 and 100 μg rHSA; andRabbit C with a mixture of 120 μg rHSA/hIL-11 fusion (equivalent of 50μg of pure bacteria-expressed IL-11), 27 μg rHSA/hEPO fusion (equivalentof 150 U of HSA-EPO determined by using the EPO-ELISA Kit, R&D SystemInc.) and 50 μg rHSA. Rabbit D was injected with 120 μg of HSA-IL-11 andabout 80 μg rHSA.

Blood samples were collected and red blood cell and platelet numberswere counted by a hemacytometer. The results are shown in FIG. 9 (panelsA, B, and C). The cell counts on the starting day of the experiment wastreated as the base line. After the treatment with the proteins, thecell counts were compared with those on the starting day and the changeswere plotted in the graphs in FIG. 9.

As shown in panel A of FIG. 9, both EPO and HSA/EPO fusion proteinstimulated the production of erythrocytes in rabbit A and C,respectively. However, in rabbit A injected with the naked EPO, thelevel of erythrocytes reached a peak around day 35 post first injectionand then declined quickly to reach near a baseline level around day 55.In contrast, in rabbit C injected with HSA/EPO fusion protein, the levelof erythrocytes increased and reached a plateau around day 35 post firstinjection but remained high till the end of the experiment. Theseresults demonstrated that HSA/EPO fusion protein has a much longerplasma half-life than the naked EPO and remains bioactive for a muchlonger time than the naked EPO in vivo. As also shown in this panel,IL-11 had little effect in stimulation of erythrocyte production inrabbit B.

As shown in panel B of FIG. 9, both IL-11 and HSA/IL-11 fusion proteinstimulated the production of platelets in rabbit B and C, respectively.However, in rabbit B injected with the naked IL-11, the level ofplatelets reached a peak around day 20 post first injection and thendeclined quickly to reach near a baseline level around day 43. Incontrast, in rabbit C injected with HSA/IL11 fusion protein, the levelof platelets increased and reached a plateau around day 40 post firstinjection but remained high till the end of the experiment. Theseresults demonstrated that HSA/IL-11 fusion protein has a much longerplasma half-life than the naked IL-11 and remains bioactive for a muchlonger time than the naked IL-11 in vivo. As also shown in this panel,EPO had little effect in stimulation of platelet production in rabbit A.

Panel C in FIG. 9 compares the effects of a combination of HSA/EPO andHSA/IL-11 with HSA/EPO and HSA/IL-11 individually in stimulation of theproduction of erythrocytes and platelets on day 35 post first injection.As shown in this panel, compared with individual HSA/EPO and HSA/IL-11,a combination of HSA/EPO and HSA/IL-11 had much stronger effects instimulating the production of both erythrocytes and platelets. Forexample, the erythrocyte level in the rabbit C which was injected with27 μg of HSA/EPO in combination 120 μg HSA/IL-11 with is higher than thetotal erythrocyte level combining that in rabbit A (injected with 10 μgHSA/EPO) and rabbit D (120 μg HSA/IL-11). Since the molar ratio of HSAto EPO in the HSA/EPO fusion is about 5:1 and the amount of HSA/EPO inrabbit C is only 2.7 folds of that in Rabbit A, it can be predicted thatif the amount of HSA/EPO is increased 5 folds so that a equal mole ofHSA/EPO in both the HSA/EPO+HSA/IL-11 combination and HSA/EPO alone isadministered, the erythrocyte level in the animal should be even higherwith the administration of the combination. Based on this analysis, itcan be reasonably inferred that a combination of HSA/EPO+HSA/IL-11fusion proteins has a synergistic effect in stimulating multiple bloodcell production.

12. Expression and Scale-Up of HSA-CPSF Fusion Protein by Fermentation

In this example, it is shown that expression and scale-up are mucheasier by using a Pichia system than other currently available systems.After Pichia recombinants were isolated, expression of both Mut+ andMut^(s) recombinants was tested. This involved growing a small cultureof each recombinant, inducing with methanol, and taking sample atdifferent time points. For secretory expression, both the cell pelletand supernatant were analyzed from each time point. The samples wereanalyzed on SDS-PAGE gels by using both Coomassie staining and Westernblot. Bioactivities of expressed samples were tested and the expressionlevels and purity were monitored in each step for production of HSAfusion proteins. FIG. 10 shows a SDS-PAGE of the purity of variousHSA-CPSFs fusion proteins.

REFERENCES CITED

U.S. Patent Documents

4,683,293 July, 1987 Craig 4,847,201 July, 1989 Kaswasaki, et al.5,292,646 March, 1994 McCoy, et al. 5,371,193 December, 1994 Bennett, etal. 5,457,038 October, 1995 Trinchieri, et al. 5,466,781 November, 1995Dorin, et al. 5,470,569 November, 1995 Kaswasaki, et al. 5,547,933August, 1996 Lin, et al. 5,686,263 November, 1997 Wurm 6,022,953February, 2000 Ralph, et al. 6,160,089 December, 2000 Honjo, et al.6,162,467 December, 2000 Baumann, et al. (*) 6,165,470 December, 2000Becquart, et al. 6,207,802 March, 2001 Zsebo, et al. 6,254,870 July,2001 Staten, et al. 6,258,559 July, 2001 Zamost 6,300,314 October, 2001Wallner, et al. 6,309,632 October, 2001 Agosti 6,322,779 November, 2001Halenbeck, et al. 6,326,198 December, 2001 Emerson, et al.Foreign Patent Documents

WO01/79271 October, 2001 Balance et al. WO01/79442 October, 2001 Rosenand HaseltinOther References

-   Testa, et al., Exp. Hematol., 8(Supp. 8), 144-152 (1980).-   Tong, et al., J. Biol. Chem., 256(24), 12666-12672 (1981).-   Goldwasser, J. Cell. Physiol., 110(Supp 1), 133-135 (1982).-   Finch, Blood, 60(6), 1241-1246 (1982);-   Sytowski, et al., Exp. Hematol., 8(Supp 8), 52-64 (1980).-   Naughton, Ann. Clin. Lab. Sci., 13(5), 432-438 (1983).-   Weiss, et al., Am. J. Vet. Res., 44(10), 1832-1835 (1983).-   Lappin, et al., Exp. Hematol., 11(7), 661-666 (1983).-   Baciu, et al., Ann. N.Y. Acad. Sci., 414, 66-72 (1983).-   Murphy, et al., Acta. Haematologica Japonica, 46(7), 1380-1396    (1983).-   Dessypris, et al., Brit. J. Haematol , 56, 295-306 (1984).-   Emmanouel, et al., Am. J. Physiol., 247 (1 Pt 2), F168-76 (1984).-   Lin, F K et al., “Cloning and expression of human erythropoietin    gene”, Proc. Natl. Acad. Sci. USA., 82(22):7580-7584 (1985).-   Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977)-   Saiki, R. K. et al, Science 230:1350-1354, 1985-   Maniatis T et al., “Molecular cloning, a Laboratory Manual”, Cold    Spring Harbor laboratory, Cold Spring Harbor, N.Y., 1982-   Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a    (Pichia manu)-   Watanabe, et al., Pharm Res 2001 December:18(12):1775.-   Kobayashi, K et al., Ther Apher, November:2(4):257-62, 1998.(Pichia)-   Buckholz and Gleeson, 1991; Cregg and Higgins, 1995 (Pichia)-   Metcalf, D., Blood, 67, 257, 1986-   Mufson, R. A. et al., Cellular Immunol., 119, 182, 1989-   Wong, G., et al., Science, 228, 810, 1986.-   Metcalf, D., Blood, 67, 257, 1986.-   Hattersley, G., et al., J. Cell Physiol., 137, 199, 1988.-   Nicola, N., Immunol. Today, 8, 134, 1987.-   Kitamura, T., et al., J. cell Physiol., 140, 323, 1989.-   Kawasaki, E. S., et al., Science, 230, 291, 1985.-   Morrison, “Bioprocessing in Space—an Overview”, pp. 557-571 in The    World Biotech REPOrt 1984, Volume 2:USA, (Online Publications, New    York, N.Y. 1984).-   Vedovato, et al., Acta. Haematol, 71, 211-213 (1984)-   Vichinsky, et al., J. Pediatr., 105(1), 15-21 (1984)-   Cotes, et al., Brit. J. Obstet. Gyneacol., 90(4), 304-311 (1983)-   Haga, et al., Acta. Pediatr. Scand., 72, 827-831 (1983)-   Claus-Walker, et al., Arch. Phys. Med. Rehabil., 65, 370-374 (1984)-   Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182 (1984)-   Miller, et al., Brit. J. Haematol., 52, 545-590 (1982)-   Udupa, et al., J. Lab. Clin. Med., 103(4), 574-580 and 581-588    (1984);-   Lipschitz, et al., Blood, 63(3), 502-509 (1983)-   Dainiak, et al., Cancer, 51(6), 1101-1106 (1983)-   Schwartz, et al., Otolaryngol., 109, 269-272 (1983)-   Pennathur-Das, et al., Blood, 63(5), 1168-71 (1984)-   Haddy, Am. Jour. Ped. Hematol./Oncol., 4, 191-196, (1982)-   Eschbach, et al. J. Clin. Invest., 74(2), pp. 434-441, (1984-   Krane, Henry Ford Hosp. Med. J., 31(3), 177-181 (1983).

1. A human serum albumin-interleukin-11 (HSA-IL-11) fusion proteincomprising: (a) the amino acid sequence set forth in SEQ ID NO: 2; (b)the amino acid sequence encoded by the polynucleotide set forth in SEQID NO: 1; or (c) the amino acid sequence encoded by the polynucleotidecontained in the yeast strain designated as YZ-HSA/IL-11 in ATCC®Deposit No: PTA-4607, said HSA-IL-11 fusion protein having a plasmahalf-life longer than a plasma half-life of interleukin-11, and saidHSA-IL-11 fusion protein being a human serum albumin-humaninterleukin-11 fusion protein, wherein the human interleukin-11 islinked directly to the C-terminus of the human serum albumin without apeptide linker, and said HSA-IL-11 fusion protein has a shelf-life fourtimes or longer than a shelf-life of the interleukin-11.
 2. TheHSA-IL-11 fusion protein of claim 1, wherein said HSA-IL-11 fusionprotein has a shelf-life ten times longer than a shelf-life of theinterleukin-11.
 3. The HSA-IL-11 fusion protein of claim 1, wherein saidHSA-IL-11 fusion protein has said plasma half-life four times or longerthan said plasma half-life of the interleukin-11.
 4. A compositioncomprising said HSA-IL-11 fusion protein of claim 1 and apharmaceutically acceptable excipient.
 5. The composition of claim 4further comprising a human serum albumin-human erythropoietin (HSA-EPO)fusion protein.